Mch3, a novel apoptotic protease, nucleic acids encoding and methods of use

ABSTRACT

The invention provides an isolated gene encoding Mch3, or functional fragment thereof. Also provided is an isolated nucleic acid sequence encoding Mch3 or functional fragment thereof. The gene or nucleic acid sequence can be single or double stranded nucleic acids corresponding to coding or non-coding strands of the Mch3 nucleotide sequence. An isolated Mch3 polypeptide or functional fragment thereof is also provided.

This patent is a divisional of U.S. patent application Ser. No.09/944,851 (filed Aug. 31, 2001) now abandoned, which, in turn, is acontinuation of U.S. patent application Ser. No. 08/556,627 (filed Nov.13, 1995) now U.S. Pat. No. 6,462,175.

This invention was made with government support under grants AI 35035-01from the National Institutes of Health. Accordingly, the government hascertain rights to this invention.

Throughout this application various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

BACKGROUND OF THE INVENTION

The present invention relates generally to apoptosis or, programed celldeath, and more particularly, to a novel cysteine protease which can beused to modulate apoptosis for the therapeutic treatment of humandiseases.

Apoptosis is a normal physiological process of cell death that plays acritical role in the regulation of tissue homeostasis by ensuring thatthe rate of new cell accumulation produced by cell division is offset bya commensurate rate of cell loss due to death. It has now become clearthat disturbances in apoptosis (also referred to as physiological celldeath or programmed cell death) that prevent or delay normal cellturnover can be just as important to the pathogenesis of diseases as areknown abnormalities in the regulation of proliferation and the cellcycle. Like cell division, which is controlled through complexinteractions between cell cycle regulatory proteins, apoptosis issimilarly regulated under normal circumstances by the interaction ofgene products that either induce or inhibit cell death.

The stimuli which regulate the function of these apoptotic gene productsinclude both extracellular and intracellular signals. Either thepresence or the removal of a particular stimuli can be sufficient toevoke a positive or negative apoptotic signal. For example,physiological stimuli that prevent or inhibit apoptosis include, forexample, growth factors, extracellular matrix, CD40 ligand, viral geneproducts neutral amino acids, zinc, estrogen and androgens. In contrast,stimuli which promote apoptosis include growth factors such as tumornecrosis factor (TNF), Fas, and transforming growth factor β (TGFβ),neurotransmitters, growth factor withdrawal, loss of extracellularmatrix attachment, intracellular calcium and glucocorticoids, forexample. Other stimuli, including those of environmental andpathogenetic origins, also exist which can either induce or inhibitprogrammed cell death. Although apoptosis is mediated by diverse signalsand complex interactions of cellular gene products, the results of theseinteractions ultimately feed into a cell death pathway that isevolutionarily conserved between humans and invertebrates.

Several gene products which modulate the apoptotic process have now beenidentified. Although these products can in general be separated into twobasic categories, gene products from each category can function toinhibit or induce programmed cell death. One family of gene products arethose which are members of the Bcl-2 family of proteins. Bcl-2, is thebest characterized member of this family and inhibits apoptosis whenoverexpressed in cells. Other members of this gene family include, forexample, Bax, Bak, Bcl-x_(L), Bcl-x_(s), and Bad. While some of theseproteins can prevent apoptosis others augment apoptosis (e.g. Bcl-x_(L)and Bak, respectively).

A second family of gene products, the interleukin-1-beta convertingenzyme (ICE) family of proteases are related genetically to the C.elegans Ced-3 gene product which was initially shown to be required forprogrammed cell death in the roundworm, C. elegans. The ICE family ofproteases includes human ICE, ICH-1_(L), ICH-1_(S), CPP32, Mch2, ICH-2and ICE_(rel)-III. Among the common features of these gene products isthat 1) they are cysteine proteases with specificity for substratecleavage at Asp-x bonds, 2) they share a conserved pentapeptide sequence(QACRG) (SEQ ID NO: 14) within the active site and 3) they aresynthesized as proenzymes that require proteolytic cleavage at specificaspartate residues for activation of protease activity. Cleavage of theproenzyme produces two polypeptide protease subunits of approximately 20kD (p20) and 10 kD (p10) which, in the case of ICE, combinenon-covalently to form a tetramer comprised of two p20:p10 heterodimers.Although these proteases, when expressed in cells, induce cell death,several alternative structural forms of these proteases, such as ICEδ,ICEε, ICH-1_(s) and Mch2β, actually function to inhibit apoptosis.

In addition to the Bcl-2 and Ced-3/ICE gene families which play a rolein apoptosis in mammalian cells, it has become increasingly apparentthat other gene products exist which are important in mammalian celldeath and which have yet to be identified. For example, in addition toCed-3, another C. elegans gene known as Ced-4 exists which is alsorequired for programmed cell death in C. elegans. However, mammalianhomologues of this protein remain elusive and have not yet beenidentified. Further, it is ambiguous as to whether other genes existwhich belong to either of the above two apoptotic gene families or whatrole they may play in the programmed cell death pathway.

As stated previously, apoptosis plays an important physiological role inmaintaining tissue homeostasis. Programmed cell death functions inphysiological processes such as embryonic development, immune cellregulation and normal cellular turnover. Therefore, the dysfunction, orloss of regulated apoptosis can lead to a variety of pathologicaldisease states. For example, the loss of apoptosis can lead to thepathological accumulation of self-reactive lymphocytes such as thatoccurring with many autoimmune diseases. Inappropriate loss of apoptosiscan also lead to the accumulation of virally infected cells and ofhyperproliferative cells such as neoplastic or tumor cells Similarly,the inappropriate activation of apoptosis can also contribute to avariety of pathological disease states including, for example, acquiredimmunodeficiency syndrome (AIDS), neurodegenerative diseases andischemic injury. Treatments which are specifically designed to modulatethe apoptotic pathways in these and other pathological conditions canchange the natural progression of many of these diseases.

Thus, there exists a need to identify new apoptotic genes and their geneproducts and for methods of modulating this process for the therapeutictreatment of human diseases. The present invention satisfies this needand provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides an isolated gene encoding Mch3, or functionalfragment thereof. Also provided is an isolated nucleic acid sequenceencoding Mch3 or functional fragment thereof. The gene or nucleic acidsequence can be single or double stranded nucleic acids corresponding tocoding or non-coding strands of the Mch3 nucleotide sequence. Anisolated Mch3 polypeptide or functional fragment thereof is alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide and predicted amino acid sequence of thehuman Mch3α of (SEQ ID NOS:1 and 2) and Mch3β (SEQ ID NOS:3 and 4),respectively. The nucleotide sequence of Mch3β that is different fromthat of Mch3α is shown below the nucleotide sequence of Mch3α. Thepredicted amino acid sequence of Mch3α is shown above the nucleotidesequence. The predicted amino acid sequence of Mch3β that is differentfrom that of Mch3α is shown below the nucleotide sequence. Dotted linesindicate the spliced sequences in Mch3α and β. The underlined Mch3αnucleotide sequence is deleted in Mch3β and is replaced by the intronicsequence shown below it. The putative active site pentapeptide of Mch3αis boxed. The putative p20, p17 and p12 cleavage sites are indicatedwith a horizontal arrow. The vertical arrow indicates an intronlocation. Amino acid and nucleotide residues are numbered to the rightof each sequence.

FIG. 2 shows Sf9 cells that were infected with the following recombinantbaculoviruses: column 1, AcNPV-Mch3α-p17; column 2, AcNPV-Mch3α-p12;column 3, AcNPV-Mch3α-p17 and AcNPV-Mch3α-p12; column 4,AcNPV-CPP32-p17; column 5, AcNPV-CPP32-p12; column 6, AcNPV-CPP32-p17and AcNPV-CPP32-p12; column 7, AcNPV-MCH3α-p17 and AcNPV-CPP32-p17;column 8, AcNPV-Mch3α-p12 and AcNPV-CPP32-p12; column 9, AcNPV-Mch3α-p17and AcNPV-CPP32-p12; column 10, AcNPV-CPP32-p17 and AcNPV-Mch3α-p12. 42h postinfection, cells were examined microscopically and several fieldswere counted (average 1500 cells/condition) and the number of apoptoticcells was expressed as a percentage of total cells counted.

FIG. 3 shows the cleavage of ProMch3α by CPP32.

(A) pbluescript vectors containing a GST-Mch3α2 or a GST-CPP32 insertsunder the T7 promoter were linearized with the appropriate restrictionenzymes as indicated by arrows and then used as templates for in vitrotranscription and translation in the presence of ³⁵S-methionine.

(B) Lanes 1 and 2, the GST-Mch3α2 DNA template was linearized with EcoRI before transcription/translation and the products of translation wereincubated with buffer (lane 1) or CPP32 (lane 2) for 30 min at 30° C.Small amount of full length GST-Mch3α2 can be seen as a 64 kDatranslation product (lane 1) or 35 kDa cleavage product due toincomplete digestion of the DNA template with Eco RI. Lanes 3-6, TheGST-Mch3α2 DNA template was linearized with Xho I beforetranscription/translation and the products of translation were incubatedfor 30 min on ice with buffer (lane 3) or at 30° C. with buffer (lane4), CPP32 (lane 5) or Mch3α (lane 6).

(C) The GST-CPP32 DNA template was linearized with Eco RI beforetranscription/translation and the products of translation were incubatedfor 30 min at 30° C. with buffer (lane 1), Mch3α (lane 2) or CPP32 (lane3).

(D) The GST-Mch3α was immobilized on a GST-Sepharose resin and theresin-GST-Mch3α2 was incubated for 1 h on ice with buffer (lane 1) orwith CPP32 (lane 2) at 30° C. The protein products in B and C wereanalyzed on a 14% SDS-gels and in D on a 10-20% gradient SDS-gels. Thearrows on the right of B and C indicate the cleavage products.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a novel apoptotic cysteine protease termedMch3. This protease is a member of the ICE family of cysteine proteasewhich includes, for example, ICE, ICH-1_(L), ICH-1_(S), CPP32, Mch2,ICH-2 and ICE_(rel) ⁻III. Similar to other ICE related proteases, Mch3is synthesized as a larger proenzyme and becomes active followingproteolytic cleavage into two subunits of approximately 17 kD (p17) and12 kD (p12). The two subunits form heterodimers which associate witheach other into an active complex. Mch3 contains no known functionallysignificant sequence identities outside of the ICE family of cysteineproteases. Similar to these other cysteine proteases, substratespecificity uniquely requires an Asp residue in the P1 position of thesubstrate binding site with a small, preferably hydrophobic, residue inthe P1′ position. Overexpression of Mch3 protease results in theinduction of apoptosis.

In one embodiment, the invention is directed to nucleic acids encodingthe apoptotic cysteine protease Mch3. The nucleic acids are used toproduce recombinant Mch3 protease, whose activity can be measuredenzymatically. The recombinant Mch3 polypeptides are used to screen forMch3 inhibitory compounds. Such pharmaceutical compounds are useful forthe treatment or prevention of diseases which are characterized byapoptotic cell death Alternatively, the Mch3 polypeptides can be used toscreen for pharmaceutical compounds which activate or act as agonists ofMch3 such as by inducing cleavage of the proenzyme into its activesubunits. Such compounds are useful for the treatment or prevention ofdiseases which are characterized by the loss of apoptotic cell death.

As used herein, the term “substantially” when referring to a Mch3nucleotide or amino acid sequence is intended to refer to the degree towhich two sequences of between about 15-30 or more nucelotides inlength, are identical or similar so as to be considered by those skilledin the art to be functionally equivalent. For example, the Mch3 nucleicacids of the invention have a nucleotide sequence substantially the sameas that shown in FIG. 1 and in SEQ ID NOS: 1 and 3. Thus, if a secondsequence is substantially the same as that shown in FIG. 1 (SEQ ID NOS:1 and 3), then it is considered functionally equivalent by those skilledin the art. Methods for sequence comparisons and determinations ofsimilarity are well known and routine within the art.

Functionally equivalent nucleic acid sequences include, for example,sequences that are related, but different and encode the same Mch3polypeptide due to the degeneracy of the genetic code as well assequences that are related, but different and encode a different Mch3polypeptide that exhibits similar functional activity. In both cases,the nucleic acids encode functionally equivalent gene products.Functional fragments of Mch3 encoding nucleic acids such asoligonucleotides, polyoligonucleotides, primers and the like are alsoconsidered to be within the definition of the term and the invention asclaimed. Functional equivalency is also relevant to Mch3 nucleic acidswhich do not encode gene products, for example, but instead arefunctional elements in and of themselves. Specific examples of suchfunctional nucleic acids include, for example, promoters, enhancers andother gene expression regulatory elements.

Mch3 polypeptides of the invention have an amino acid sequencesubstantially similar to that shown in FIG. 1 and in SEQ ID NOS:2 and 4.Functionally equivalent Mch3 amino acid sequences similarly includes,for example, related, but different sequences so long as the differentpolypeptide exhibits at least one functional activity of Mch3. Suchrelated, but different polypeptides include, for example, substitutionsof conserved and non-essential amino acids. Fragments and functionaldomains of Mch3 are similarly included within the definition of the termand the claimed invention.

Therefore, it is understood that limited modifications may be madewithout destroying the biological function of the Mch3 polypeptide andthat only a portion of the entire primary structure may be required inorder to effect activity. For example, minor modifications of the Mch3amino acid sequences (SEQ ID NOS: 2 and 4) which do not destroy theiractivity also fall within the definition of Mch3 and within thedefinition of the polypeptide claimed as such. Also, for example,genetically engineered fragments of Mch3 either alone or fused toheterologous proteins such as fusion proteins, for example, that retainmeasurable enzymatic activity fall within the definition of thepolypeptides claimed as such. It is understood that minor modificationsof primary amino acid sequence may result in polypeptides which havesubstantially equivalent or enhanced function as compared to thesequence set forth in FIG. 1 (SEQ ID NOS 2 and 4). These modificationsmay be deliberate, as through site-directed mutagenesis, or may beaccidental such as through mutation in hosts which are Mch3 producers.All of these modifications are included as long as Mch3 biologicalfunction is retained. Further, various molecules can be attached toMch3, for example, other proteins, carbohydrates, lipids, or chemicalmoieties. Such modifications are included within the definition of Mch3polypeptides.

The invention provides a gene encoding Mch3, or fragment thereof. Theinvention also provides an isolated nucleic acid sequence encoding Mch3,or fragment thereof. The gene and nucleic acid sequences encodesubstantially the sequence as shown in SEQ ID NOS:1 and 3. Fragments ofthe gene or nucleic acid sequence are provided which comprise single ordouble stranded nucleic acids having substantially the sequences shownin SEQ ID NOS:1 and 3.

The Mch3 nucleic acids of the present invention were identified andisolated by a novel approach of searching a human database of expressedsequence tags (ESTs) under various stringencies to identify potentialnew sequence fragments which may have homology to the ICE family ofcysteine proteases. Novel sequences identified as having potentialhomology to the ICE family of apoptotic proteases can be used to designprimers for attempting PCR amplification. The second primer is designedto encompass homologous regions in nucleic acid sequences that encodeknown ICE protease family members. In this specific case, the primer wasdirected to the GSWFI/GSWYI (SEQ ID NOS: 12-13) pentapeptide sequencethat is conserved in a number of the ICE/Ced-3 family of proteases. Theprimer design should take into account the predicted strandedness ofboth the EST sequence primer and the known primer. Thus, only if thehomology search and hybridization conditions are successfullydetermined, will such an approach allow PCR amplification of a fragmentof the putative novel protease cDNA. As searching a genetic data basewill yield homologous sequence matches to any query nucleotide sequence,additional criteria must be used to identify the authentic ICE familyhomologue from among the non-specific homology matches. ICE familymembers share the highest degree of homology in the active site andcatalytically important amino acid residues. A given EST returned by thesearch may not include one of these highly homologous sites, but rather,may only include a region within the protease with cryptic homology.Confirming an EST as a novel ICE protease involves translation of allthe positive EST hits in three different reading frames and subsequentidentification of conservative active site or catalytically importantamino acid sequence motifs. Then, using conventional cDNA cloning, afull length cDNA of the putative novel protease can be obtained and 1)analyzed for overall structural homology to ICE family members, 2)recombinantly expressed and analyzed for cysteine protease activity, and3) analyzed for the induction of programmed cell death by heterologousexpression of the cDNA in appropriate cells.

Alternative methods than that described above for isolating Mch3encoding nucleic acids can similarly be employed. For example, using theteachings described herein, those skilled in the art can routinelyisolate and manipulate Mch3 nucleic acids using methods well known inthe art. All that is necessary is the sequence of the Mch3 encodingnucleic acids (FIG. 1 and SEQ ID NOS:1 and 3) or the Mch3 amino acidsequence (FIG. 1 and SEQ ID NOS:2 and 4). Such methods include, forexample, screening a cDNA or genomic library by using syntheticoligonucleotides, nucleic acid fragments or primers as hybridizationprobes. Alternatively, antibodies to the Mch3 amino acid sequence orfragments thereof can be generated and used to screen an expressionlibrary to isolate Mch3 encoding nucleic acids. Other binding reagentsto Mch3 polypeptides can similarly be used to isolate Mch3 polypeptideshaving substantially the amino acid sequence show in FIG. 1. Similarly,substrate reagents such as non-cleavable peptide analogues of cysteineproteases can be used to screen and isolate Mch3 polypeptides.

In addition, recombinant DNA methods currently used by those skilled inthe art include the polymerase chain reaction (PCR) which, combined withthe Mch3 nucleotide and amino acid sequences described herein, allowseasy reproduction of Mch3 encoding sequences. Desired sequences can beamplified exponentially starting from as little as a single gene copy bymeans of PCR. The PCR technology is the subject matter of U.S. Pat. Nos.4,683,195, 4,800,159, 4,754,065, and 4,683,202 all of which areincorporated by reference herein.

The above described methods are known to those skilled in the art andare described, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992) andthe various references cited therein and in Ansubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989); and in Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York (1989). These references and thepublications cited therein are hereby expressly incorporated herein byreference.

The invention provides an isolated Mch3 polypeptide comprisingsubstantially the amino acid sequence as that shown in FIG. 1 (SEQ IDNOS:2 and 4). Mch3 functional fragments are also provided. A specificexample of an Mch3 functional fragment is the catalytic domain whichcontains the active site amino acid sequence QACRG.

Isolated Mch3 polypeptides of the invention can be obtained by a varietyof methods known within the art. For example, the isolated peptides canbe purified by biochemical methods including, for example, affinitychromatography. Affinity matrices which can be used for Mch3 isolationcan be anti-Mch3 monoclonal or polyclonal antibodies prepared againstthe sequence shown in FIG. 1 (SEQ ID NOS:2 and 4), or fragments thereofsuch as synthetic peptides. Alternatively, substrate analogues orenzymatic inhibitors of Mch3 can similarly be used as affinity matricesto isolate substantially pure Mch3 polypeptides of the invention.

Mch3 polypeptides can also be produced by recombinant methods known tothose skilled in the art. Recombinant Mch3 polypeptides include, forexample, an amino acid sequence substantially the same as that shown inFIG. 1 (SEQ ID NOS:2 and 4) as well as fusion proteins and fragmentsthereof. The Mch3 encoding nucleic acids can be cloned into theappropriate vectors for propagation, manipulation and expression. Suchvectors are known or can be constructed by those skilled in the art andshould contain all expression elements necessary for the transcription,translation, regulation, and if desired, sorting of the Mch3polypeptides. The vectors can also be for use in either procaryotic oreucaryotic host systems so long as the expression and regulatoryelements are of compatible origin. One of ordinary skill in the art willknow which host systems are compatible with a particular vector. Therecombinant polypeptides produced can be isolated by the methodsdescribed above.

Apoptosis plays a significant role in numerous pathological conditionsin that programed cell death is either inhibited, resulting in increasedcell survival, or enhanced which results in the loss of cell viability.Examples of pathological conditions resulting from increased cellsurvival include cancers such as lymphomas, carcinomas and hormonedependent tumors. Such hormone dependent tumors include, for example,breast, prostrate and ovarian cancer. Autoimmune diseases such assystemic lupus erythematosus and immune-mediated glomerulonephritis aswell as viral infections such as herpesvirus, poxvirus and adenovirusalso result from increased cell survival or the inhibition of apoptosis.

In contrast, apoptotic diseases where enhanced programed cell death is aprevalent cause generally includes, for example, degenerative disorderssuch as Alzheimer's disease, Parkinson's disease, Amyotrophic lateralsclerosis, Retinitis pigmentosa, and Cerebellar degeneration. Otherdiseases associated with increased apoptosis include, for example,myelodysplastic syndromes such as aplastic anemia and ischemic injuryincluding myocardial infarction, stroke and reperfusion injury.

The Mch3 encoding nucleic acids and polypeptides of the invention can beused to diagnose, treat or reduce the severity of cell death mediateddiseases such as those described above as well as other diseasesmediated by either increased or decreased programmed cell death.Additionally, the Mch3 encoding nucleic acids and polypeptides of theinvention can be used to screen for pharmaceutical compounds andmacromolecules which inhibit or promote Mch3 mediated apoptosis.

For example, the Mch3 encoding nucleic acids, polypeptides andfunctional fragments thereof can be used to diagnose, or to generatereagents to diagnose diseases mediated or characterized by programedcell death. Diagnosis can be by nucleic acid probe hybridization withMch3 containing nucleotide sequences, antibody or ligand mediateddetection with Mch3 binding agents or by enzyme catalysis of detectableMch3 substrates. Such methods are routine to those skilled in the art.Detection can be performed ex vivo, for example, by removing a cell ortissue sample from an individual exhibiting or suspected of exhibiting acell death mediated disease. Correlation of increased Mch3 expression oractivity is indicative of diseases characterized by enhanced programmedcell death whereas correlation of decreased Mch3 expression or activityis indicative of diseases characterized by the inhibition of programmedcell death.

The above Mch3 polypeptides can also be formulated into pharmaceuticalcompositions known within the art for the treatment of cell deathmediated diseases characterized by increased cell survival andproliferation. Functional fragments and peptides such as the catalyticdomain of Mch3 can similarly be formulated for the treatment of suchdiseases associated with increased cell survival and proliferation.Administration of Mch3 polypeptides and functional fragments thereofwill induce apoptosis in treated cells and eliminate those cellscharacterized by increased cell survival or proliferation.Administration of non-Mch3 polypeptides that do not directly act on Mch3substrates but induce the activation of the Mch3 protease can similarlybe used for the treatment of diseases characterized by increased cellsurvival and proliferation.

To be effective, the Mch3 polypeptides must be introduced into the cellscharacterized by increased cell survival. Introduction can beaccomplished by a variety of means known within the art including, forexample, lipid vesicles and receptor mediated endocytosis. Targeting tothe appropriate cell type can similarly be accomplished throughconjugation to specific receptor ligands, specific target cellantibodies and the like.

The Mch3 polypeptides are administered by conventional methods, indosages which are sufficient to induce apoptosis in the cellscharacterized by increased cell survival or proliferation. Such dosagesare known or can be easily determined by those skilled in the art.Administration can be accomplished by, for example, intravenous,interperitonal or subcutaneous injection. Administration can beperformed in a variety of different regimes which include single highdose administration or repeated small dose administration or acombination of both. The dosing will depend on the cell type,progression of the disease and overall health of the individual and willbe known or can be determined by those skilled in the art.

In contrast to the induction of Mch3 mediated apoptosis for thetreatment of pathological conditions characterized by increased cellsurvival or proliferation, inhibitors of Mch3 can be used to treatdiseases characterized by increased programmed cell death. Suchinhibitors can be, for example, anti-Mch3 antibodies, proteins, or smallpeptidyl protease inhibitors which are formulated in a medium whichallows introduction into the desired cell type. Alternatively, suchinhibitors can be attached to targeting ligands for introduction by cellmediated endocytosis and other receptor mediated events. Specificexamples of Mch3 peptidyl inhibitors are described in Table I of ExampleII and includes suicide inhibitors and substrate analogues such as thetetrapeptide DEVD aldehyde (SEQ ID NO: 15), YVAD aldehyde (SEQ ID NO:16) and the cowpox virus protein Crm A, for example. Other inhibitors ofMch3 include, for example, small molecules and organic compounds whichbind and inactivate Mch3 by a competitive or non-competitive typemechanism. Molecules or compounds which indirectly inhibit the Mch3pathway can also be used as inhibitors of Mch3. Mch3 inhibitors can beidentified by screening for molecules which demonstrate specific orbeneficial Mch3 inhibitory activity. Such methods are described furtherbelow and can be practiced by those skilled in the art given the Mch3nucleotide and amino acid sequences described herein.

Dominant/negative inhibitors of Mch3 can also be used to treat or reducethe severity of diseases characterized by increased programmed celldeath. In this regard, Mch3β polypeptides which lack the active siteQACRG (SEQ ID NO: 14) can be used to bind p12 subunits of Mch3 andprevent active tetrameric complexes from forming. The mechanism of Mch3βdominant inhibition of Mch3α is indicated to be similar to the dominantnegative inhibition of Ich-1_(L) by Ich-1_(s). Subunits from other ICErelated cysteine proteases can similarly be used as dominant/negativeinhibitors of Mch3 activity and therefore treat diseases mediated byprogrammed cell death. Such subunits should be selected so that theybind either the p17 or p12 Mch3 polypeptides and prevent their assemblyinto active tetrameric protease complexes. Moreover, Mch3 subunits whichhave been modified so as to be catalytically inactive can also be usedas dominant negative inhibitors of Mch3. Such modifications include, forexample, mutation of the active site cysteine residue to include but notlimited to Alanine or glycine.

Mch3 substrate antagonists can similarly be used to treat or reduce theseverity of diseases mediated by increased programmed cell death. Suchsubstrate antagonists can bind to and inhibit cleavage by Mch3.Inhibition of substrate cleavage prevents commitment progression ofprogrammed cell death. Substrate antagonists include, for example,ligands and small molecule compounds.

Treatment or reduction of the severity of cell death mediated diseasescan also be accomplished by introducing expressible nucleic acidsencoding Mch3 polypeptides or functional fragments thereof into cellscharacterized by such diseases. For example, elevated synthesis rates ofMch3 can be achieved by, for example, using recombinant expressionvectors and gene transfer technology. Such methods are well known withinthe art and will be described below with reference to recombinant viralvectors. Other vectors compatible with the appropriate targeted cell canaccomplish the same goal and therefore can be substituted in the methodsdescribed herein in place of recombinant viral vectors.

Recombinant viral vectors are useful for in vivo expression of a desirednucleic acid because they offer advantages such as lateral infection andtargeting specificity. Lateral infection is inherent in the lifecycleof, for example, retroviruses and is the process by which a singleinfected cell produces many progeny virions that bud off and infectneighboring cells. The result is a large area becomes rapidly infected,most of which were not initially infected by the original viralparticles. This is in contrast to vertical-type of infection in whichthe infectious agent spreads only through daughter progeny. Viralvectors can also be produced that are unable to spread laterally. Thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

Typically, viruses infect and propagate in specific cell types.Therefore, the targeting specificity of viral vectors utilizes thisnatural specificity to in turn specifically introduce a desired geneinto predetermined cell types. The vector to be used in the methods ofthe invention will depend on desired cell type to be targeted. Forexample, if neurodegenerative diseases are to be treated by decreasingthe Mch3 activity of affected neuronal cells then a vector specific forcells of the neuronal cell lineage should be used. Likewise, if diseasesor pathological conditions of the hematopoietic system are to betreated, than a viral vector that is specific for blood cells and theirprecursors, preferably for the specific type of hematopoietic cell,should be used. Moreover, such vectors can additionally be modified withspecific receptors or ligands and the like to modify or alter targetspecificity through receptor mediated events. These modificationprocedures can be performed by, for example, recombinant DNA techniquesor synthetic chemistry procedures. The specific type of vector willdepend upon the intended application. The actual vectors are also knownand readily available within the art or can be constructed by oneskilled in the art using well known methodology.

Viral vectors encoding Mch3 nucleic acids or inhibitors of Mch3 can beadministered in several ways to obtain expression of such sequences andtherefore either increase or decrease the activity of Mch3 in the cellsaffected by the disease or pathological condition. If viral vectors areused, for example, the procedure can take advantage of their targetspecificity and consequently, do not have to be administered locally atthe diseased site. However, local administration can provide a quickerand more effective treatment. Administration can also be performed by,for example, intravenous or subcutaneous injection into the subject.Injection of the viral vectors into the spinal fluid can also be used asa mode of administration, especially in the case of neurodegenerativediseases. Following injection, the viral vectors will circulate untilthey recognize host cells with the appropriate target specificity forinfection.

As described above, one mode of administration of Mch3 encoding vectorscan be by direct inoculation locally at the site of the disease orpathological condition. Local administration is advantageous becausethere is no dilution effect and therefore a smaller dose is required toachieve Mch3 expression in a majority of the targeted cells.Additionally, local inoculation can alleviate the targeting requirementrequired with other forms of administration since a vector can be usedthat infects all cells in the inoculated area. If expression is desiredin only a specific subset of cells within the inoculated area thenpromoter and expression elements that are specific for the desiredsubset can be used to accomplish this goal. Such non-targeting vectorscan be, for example, viral vectors, viral genomes, plasmids, phagemidsand the like. Transfection vehicles such as liposomes can be used tointroduce the non-viral vectors described above into recipient cellswithin the inoculated area Such transfection vehicles are known by oneskilled within the art. Alternatively, however, non-targeting vectorscan be administered directly into a tissue of any individual. Suchmethods are known within the art and are described by, for example,Wolff et al. (Science 247:1465-1468 (1990)).

Additional features can be added to the vectors to ensure safety and/orenhance therapeutic efficacy. Such features include, for example,markers that can be used to negatively select against cells infectedwith the recombinant virus. An example of such a negative selectionmarker is the TK gene described above that confers sensitivity to theantibiotic gancyclovir. Negative selection is therefore a means by whichinfection can be controlled because it provides inducible suicidethrough the addition of antibiotic. Such protection ensures that if, forexample, mutations arise that produce mutant forms of Mch3, dysfunctionof apoptosis will not occur.

As described previously, the Mch3 encoding nucleic acids and Mch3polypeptides of the invention can be used to screen for compounds whichinhibit or enhance the expression of Mch3 protease activity. Suchscreening methods are known to those skilled in the art and can beperformed by either in vitro or in vivo procedures. For example,described in Example II is a specific in vitro assay for Mch3 activity.This assay employs Mch3 polypeptide expressed in an active, processedform recombinantly in E. coli, whose protease activity is measured byincubation with a fluorescent substrate (DEVD-AMC). Also describedtherein are peptide and polypeptide inhibitors of Mch3. This assay canbe used to screen synthetic or naturally occurring compound libraries,including macromolecules, for agents which either inhibit or enhanceMch3 activity. The Mch3 polypeptides to be used in the assay can beobtained by, for example, in vitro translation, recombinant expressionor biochemical procedures. Methods other than that described in ExampleII can also be used to screen and identify compounds which inhibit Mch3.A specific example is phage display peptide libraries where greater than10⁸ peptide sequences can be screened in a single round of panning. Suchmethods as well as others are known within the art and can be utilizedto identify compounds which inhibit or enhance Mch3 activity.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Cloning and Characterization of Mch3

This Example shows the cloning, sequence analysis and tissuedistribution of Mch3. The results described herein indicate that Mch3 isa novel member of the ICE family of cysteine proteases.

To identify potentially novel members of the ICE family of cysteineproteases, an approach combining information from the GenBank databaseof human expressed sequence tags (ESTs) and PCR was employed. Initially,Ced-3/ICE-like apoptotic cysteine proteases from Jurkat T-lymphocyteswere enriched by amplification of a human Jurkat cDNA library usingdegenerate PCR primers encoding the conserved GSWFI/GSWYI (SEQ ID NO:12-13) pentapeptides (Fernandes-Alnemri et al., Cancer Res. 55:2737-2742(1995)). This amino acid sequence has been found to be conserved amongICE family members. Briefly, a 10 μl aliquot of human Jurkat λ Uni-Zap™XR cDNA library containing approximately 10⁸ pfu was denatured at 99° C.for 5 min. and used as a substrate for PCR amplification with adegenerate primer encoding the pentapeptide GSWFI/GSWYI (SEQ ID NO:12-13) and a T3 vector-specific primer (Stratagene).

The enriched library was then amplified with a primer derived from anEST sequence (T50828) identified in a homology search of the GenBankdatabase using a query nucleotide sequence corresponding to the CPP32cDNA sequence minus the untranslated nucleotides (e.g. CPP32 codingsequence). This secondary amplification was performed starting with a 10μl aliquot of the above amplified sequences combined with a primerderived from the GenBank sequence T50828 (primer T50-pr1:CCGTGGAATAGGCGAAGAG, SEQ ID NO: 5) and a second vector specific primer(SK-Zap: CAGGAATTCGGCACGAG, SEQ ID NO: 6). The secondary amplificationproducts were cloned into a Sma I cut pBluescript II KS⁺ vector. Allclones were screened by PCR using a degenerate oligonucleotidecorresponding to the conserved active site amino acid sequence QACRG andthe SK-Zap primer. Clones that were positive for the presence of theQACRG coding sequence were then subjected to DNA sequencing using T3 andT7 sequencing primers (Stratagene). This amplification and screenresulted in the identification of a Ced-3/ICE-like partial cDNA withhigh homology to CPP32 and Ced-3. The partial cDNA was then excised fromthe vector, radiolabeled and used to screen the original Jurkat λUni-Zap™ XR cDNA library. Positive λ clones were purified, rescued intothe pBluescript II SK⁻ plasmid vector and sequenced.

The second screen of the Jurkat X Uni-Zap™ XR cDNA library resulted inthe isolation of several cDNA clones. One cDNA, named Mch3, contains anopen reading frame of 909 bp that encodes a 303 amino acid protein witha predicted molecular mass of approximately 34 kD (FIG. 1 and SEQ IDNOS: 1 and 2). The initiator methionine at nucleotide 44 conforms to theconsensus Kozak translation initiation sequence (20). A second cDNAclone named Mch3β (SEQ ID NO: 3) was also identified and found tocontain a deletion and insertion corresponding to nucleotides 488-592(amino acids 149-183) of the Mch3α sequence (FIG. 1) (SEQ ID NO: 1).Mch3α also has a longer 5′ nontranslated sequence.

Exon/Intron analysis of the Mch3 genomic region that correspond to thedeletion/insertion in Mch3β revealed that Mch3β mRNA resulted from twosimultaneous alternative splicing events. The first event caused thedeletion of nucleotides 488-592 of the Mch3of sequence (SEQ ID NO: 1)due to the use of an alternative splice donor located within the codingregion of the 5′ exon and an alternative splice acceptor located withinthe 3′ intron. The second splicing event caused an insertion of 74 bpintronic sequence due to the use of an alternative spice donor locatedwithin the intron and the normal splice acceptor of the 3′ exon. All thealternative splice donor/acceptor sites used in these events conform tothe GT/AG rule. As a result of the deletion and insertion, Mch3β cDNAdid not maintain the same reading frame as Mch3α after amino acid 148.The new reading frame in Mch3β does not encode a QACRG pentapeptidesequence and it terminates with a TGA stop codon corresponding to bp837-839 of Mch3β (FIG. 1; SEQ ID NO: 1). Mch3β encodes a protein of 253amino acids with a predicted molecular mass of ˜28 kDa (SEQ ID NO: 4).

In vitro translated Mch3α and Mch35 migrate as 36 and 33 kDA proteinproducts. The smaller translation products seen in the Mch3α and Mch3βtranslation reactions are probably internally translated products.Although the calculated molecular mass of Mch3β is ˜28 kDA, itsmigration as a 33 kDa indicates posttranslational modification such asphosphorylation. This result is evident from the high number of serineresidues in Mch3β sequence that is different from Mch3α and itsmigration in SDS gels as a fuzzy band rather than a sharp distinct band.The function and activity of Mch3α is discussed further below. However,similar to the alternatively spliced Ich-1 isoform (Ich-1s), (Wang etal., Cell 78:739-750 (1994)) Mch3β is thought to be a negative regulatorof apoptosis and could inhibit the activity of the parental enzyme byacting as a dominant inhibitor.

Sequence comparison of the predicted full length Mch3α protein sequenceshows the highest homology to human CPP32 and Mch2α and the C. elegansCED-3 protein. (Fernandes-Alnemri et al., J. Biol. Chem. 269:30761-30764(1994))-Overall, Mch3α protein shares ˜53% identity (67% similarity)with CPP32, ˜35% identity (56% similarity) with Mch2α and ˜33% identity(55% similarity) with CED-3. Mch3α shows less than 30% identity withother family members such as ICE, NEDD/ICH-1, Tx (ICH-2, ICE_(rel)-II)or ICE_(rel)III. In addition to the conservation of the active siteQACRG (SEQ ID NO: 14) pentapeptide the predicted structure of Mch3αappears to be similar to CPP32. CPP32 is cleaved at Asp28 and Asp175 togenerate two polypeptides of molecular masses of 17 kDa (p17) and 12 kDa(p12) that form the active CPP32 enzyme complex. Based on the highhomology between Mch3α and CPP32, it is likely that the cleavage sitesin Mch3α are Asp53 and Asp198 (FIG. 1). Cleavage at these sites wouldgenerate two polypeptides equivalent to the p17 and p12 subunits ofCPP32. However, there are three potential aspartic acid cleavage sitesat positions 15, 20 and 23 that could be used to remove a shortpropeptide during processing of Mch3α to the active enzyme. In fact, thetetrapeptide DSVD (amino acids 20-23 of Mch3α) (SEQ ID NO: 17) is verysimilar to the DEVD (SEQ ID NO: 15) tetrapeptide substrate of CPP32.This result indicates that the Mch3α is a substrate for CPP32. Inaddition, three Asp cleavage sites (Asp193, Asp204 and Asp206) locatedbetween the two subunits may serve as potential processing sites toseparate the two subunits.

To determine if Mch3 exhibits apoptotic activity, we investigatedwhether this gene product induces early apoptosis in Sf9 baculoviruscells. Briefly, Sf9 cells were infected with recombinant baculovirusesencoding full length Mch3α, full length CPP32, or truncated Mch3α orCPP32 variants, separately or in various combinations. Cells were thenexamined microscopically for morphological signs of apoptosis such asblebbing of the cytoplasmic membrane, condensation of nuclear chromatinand release of small apoptotic bodies. In addition the genomic DNA wasexamined for internucleosomal DNA cleavage.

For the construction of transfer vectors and recombinant baculoviruses,the Mch3 cDNA was amplified by PCR using primers T50-pr3GCCATAAACTCTTCCTCACTT (SEQ ID NO: 7) and T50-pr4 ATGGCAGATCATCAGGGC (SEQID NO: 8) and subcloned into the pBluescript II SK⁻ vector. The Mch3sequence was then excised with Bam HI and subcloned into a Bam HI cutpVL1393 (Invitrogen, San Diego, Calif.) to generate the pVL-Mch3αtransfer vector. The cDNA encoding the p20 and p12 subunits of Mch3 wereamplified with PCR using the following primers (p20 subunit; T50-pr4(SEQ ID NO: 8) and Mch3-p20-TAG-CTAGTCGGCCTGGATGCCATC (SEQ ID NO: 9) andp12 subunit; Mch3-p12-ATG ATGTCGGGGCCCATCAATGAC (SEQ ID NO: 10)) andT50-pr9 GACCCATTGCTTCTCAGC (SEQ ID NO: 11)). The PCR products were thencloned into a Sma I cut pVL1393 to generate pVL-Mch3-p20 andpVL-Mch3-p12 transfer vectors. The recombinant transfer vectors werethen used to generate recombinant Baculoviruses as previously described(Summers et al., “Manual of Methods for Baculovirus Vectors and InsertCulture Procedures,” Texas Experimental Station Bulletin No, 1555 (TexasA&M University, College Station, Tex. (1987); and Alnemri et al., J.Biol. Chem. 266:3925-3936 (1991)).

For the induction of apoptosis in Sf9 cells by Mch3α and CPP32.Internucleosomal DNA cleavage was assessed as a characteristic marker.Briefly, total cellular DNA was isolated at 42 h postinfection from Sf9cells infected with the wild type baculovirus or the recombinantbaculoviruses AcNPV-Mch3α or AcNPV-ICE, which have been describedpreviously (Summers et al. and Alnemri et al. supra). The DNA sampleswere analyzed by electrophoresis in a 1.8% agarose gel containingethidium bromide.

Expression of full length Mch3α in Sf9 cells caused approximately 50% ofthe cells to undergo apoptosis by 48 h postinfection which was alsomanifested by induction of internucleosomal DNA cleavage. This result isconsistent with Mch3 being an apoptotic protease since ICE, CPP32 andMch2α yield similar results. On the other hand, truncated Mch3α (aminoacids 1-198) that encodes only the p20 subunit or truncated Mch3α (aminoacids 199-303) that encodes only the p12 subunit, were unable to induceapoptosis in Sf9 cells when expressed separately (FIG. 2, columns 1 and2). However, when these two subunits were coexpressed, ˜49% of the cellsdied by apoptosis (column 3). Similarly, the two subunits of CPP32 werenot apoptotic when expressed separately (columns 4 and 5) but wereapoptotic when coexpressed together (column 6). The most interestingresults were obtained when Mch3-p20 subunit was coexpressed withCPP32-p12 subunit or vice versa (i.e. CPP32-p20 with Mch3-p12). Thesecombinations were able to cause apoptosis in more than 50% of the cells(columns 9 and 10). No significant induction of apoptosis was observedin control cells coexpressing Mch3-p20 and CPP32-p20 together or cellscoexpressing Mch3-p12 and CPP32-p12 together (columns 7 and 8). Thesedata indicate that Mch3α and CPP32 can heterodimerize in vivo ineucaryotic cells to form active apoptotic complexes. Such a dimerizationnow increases the complexity of the apoptotic response in mammaliancells. One interesting observation so far is that all known mammalianCed3/ICE-like cysteine proteases are expressed in a single cell linenamely human Jurkat T-lymphocytes. The ability of different members ofthe ICE-family such as Mch3 and CPP32 or ICE and Tx to heterodimerizeindicates that there may be some overlap in function or subtledifferences in specifications that have yet to be characterized.

To further characterize Mch3, the tissue distribution was analyzed byNorthern blot analysis of poly A+ RNA isolated from different humantissues. The analysis was performed on Northern blots prepared byClontech containing 2 μg/lane of poly A+ RNA. Radioactive riboprobe ofMch3α was prepared using a Sma I linearized pBluescript II SK⁻-Mch3α asa substrate for T7 RNA polymerase in the presence of [α³²P] UTP. Theblot was hybridized, washed and then visualized by autoradiography. Theresults indicate a major 2.4 Kb Mch3 message was detectable in alltissues examined. The lowest expression of Mch3 mRNA was seen in wholebrain. Examination of Mch3 mRNA in different regions of the brain alsoshowed low but detectable expression. Similar tissue distribution wasalso seen with CPP32 mRNA, although the CPP32 message is more abundantthan Mch3 message in brain tissues. The size of Mch3 mRNA was consistentwith the length of the cloned Mch3α and β cDNAs (FIG. 1; SEQ ID NOS: 1and 3). Two less abundant messages of (0.8 and 3.3 Kb) were alsodetectable in some tissues such as the small intestine. The largermessage could be an incompletely processed Mch3 RNA or an alternativelyspliced Mch3 isoform. The smaller message could be a degradation productor an alternatively spliced Mch3 isoform.

The enzymatic activity of Mch3α was also characterized in vitro. Mch3was expressed in E. coli as a fusion protein with glutathioneS-transferase (GST) as described for Mch2α, ICE and CPP32 (See forexample, Alnemri et al., J. Biol. Chem. 270:4312-4317 (1995)). TwoGST-Mch3α expression vectors were constructed and transformed into DH5αbacteria. The Mch3α1 cDNAs were subcloned in-frame into the Bam HI siteof the bacterial expression vector pGEX-2T (Pharmacia, Biotech, Inc.)The first construct (Mch3al) contains a PCR generated cDNA that encodesamino acids 1-303 of Mch3α fused to the C-terminus of GST. The secondconstruct (Mch3α2) contains a Bam HI fragment derived from the Mch3 λlibrary clone that encodes the full length Mch3α and an extra 16 aminoacids derived from the 5′ nontranslated region fused to the C-terminusof GST. After induction with IPTG, bacterial extracts were prepared fromE. coli expressing the recombinant fusion proteins. The extracts wereadsorbed to glutathione-Sepharose resin, washed several times and thenanalyzed by SDS-PAGE.

The Mch3α1 preparation contained a major GST-fusion protein thatmigrated as a ˜30 kDa band. On the other hand, the Mch3α2 preparationcontains a major GST-fusion protein that migrated as a ˜32 kDa band. TheGST nonfusion protein control migrated as a ˜28 kDa protein Theseresults are consistent with autocatalytic processing and cleavage ofGST-Mch3α in bacteria most probably at Asp23 of Mch3α to generate the 30and 32 kDa GST-prodomain fusion. A minor GST-fusion protein thatmigrated as a 33 kDa band in Mch3α preparation and as a 35 kDa proteinin Mch3α2 preparation was also seen above the major 30 and 32 kDa bands,respectively. These two bands are intermediate cleavage productsgenerated by cleavage at a site C-terminal to Asp23 of Mch3α. Thisindicates that the final product of Mch3α processing is cleaved at anAsp site C-terminal to Asp23 and is likely to be Asp53.

EXAMPLE II Kinetic Properties and Enzymatic Activity of Mch3α

This Example characterizes the protease activity and substratespecificity of the apoptotic cysteine protease Mch3.

The kinetic properties of the bacterially expressed recombinant Mch3αand CPP32 were determined using the tetrapeptide substate DEVD-AMC in acontinuous fluorometric assay. The DEVD-AMC substrate is thepoly(ADP-ribose)polymerase (PARP) cleavage site P1-P4 tetrapeptide(Nicholson et al., Nature 376:37-43 (1995)). Briefly, activity of Mch3αand CPP32 was measured using bacterial lysates in ICE buffer (25 mMHEPES, 1 mM EDTA, 5 mM DTT, 0.10% CHAPS, 10% sucrose, pH 7.5) at roomtemperature (24-25° C.). K_(i)'s were determined from the hydrolysisrate of 50 μM DEVDamc (10 μM for CPP32) in ICE buffer following a 30 minpreincubation of the enzyme with inhibitor. Prior to incubation withenzyme, purified crmA was activated by incubation with 5 mM DTT for 10min at 37° C.

TABLE I Protease Parameter Mch3 CPP32 k_(cat)/K_(m) (DEVDamc, mM⁻¹s⁻¹)11 1600 K_(m) (DEVDamc, μM) 51 13 k_(cat)/K_(m) (YVADamc, mM⁻¹s⁻¹) NA0.067 Km (YVADamc, μM) NA >500 K_(i) (DEVDaldehyde, nM) 1.8 0.59 K_(i)(YVADaldehyde, μM) >10 8.5 K_(i) (CrmA, μM) >1 0.56

Both Mch3α and CPP32 exhibited a Michaelis-Menton kinetics in cleavingthis substrate with K_(m) values of 51 and 13 μM, respectively (TableI). These K_(m) values and other kinetic parameters are shown above inTable I. For example, the K_(m) value of recombinant CPP32 (13 μM) wascomparable to the K_(m) value of purified human CPP32 (9.7±1.0 μM)reported recently (Nicholson et al., supra). The peptide aldehydeDEVD-CHO was also a potent inhibitor of both Mch3α and CPP32 at low nMconcentrations (K_(iMCh3)=1.8 nM and K_(iCPP32)=0.59 nM). In contrast,the ICE inhibitor peptide aldehyde YVAD-CHO (K_(iICE)=0.76 nM) was avery weak inhibitor of both Mch3α and CPP32 (K_(iMCh3)>10 μM andK_(iCPP32)=8.5 μM). The ICE inhibitor cowpox serpin, Crm A, similarlywas also a very weak inhibitor of Mch3α and CPP32 (K_(iMch3)>1 μM andK_(iCPP32)=0.56 μM). These data indicate that the two enzymes, Mch3α andCPP32 have similar substrate specificity.

In addition, the high concentration of Crm A required to inhibit eitherCPP32 or Mch3 indicates that the target of Crm A inhibition in apoptosisis unlikely to be CPP32 or Mch3α. Therefore, Crm A inhibition ofapoptosis is likely mediated through ICE or an ICE-related protease andnot through Mch3 or CPP32. CPP32 has also been recently reported to bethe PARP cleaving enzyme in apoptosis (Nicholson et al., supra, andTewavi et al., Cell 81:1-9 (1995)). However, since our data indicatethat Mch3α has a similar substrate specificity towards PARP as CPP32 itis possible that some of this previously reported activity is due toMch3α. For example, incubation of purified bovine PARP or human Helanuclei with Mch3α resulted in a complete cleavage of PARP in less than15 min. A similar activity was also observed with CPP32 and with S/Mextracts derived from chicken DU249 cells committed to apoptosis.

Inhibition studies with the serine proteases TLCK and TPCK(N-Tosyl-L-Lysyl chloromethylketone and N-Tosyl-L-phenylalanylchloromethylketone, respectfully) revealed interesting results. At 1 mMDTT concentration TPCK was able to inhibit both Mch3α and CPP32 PARPcleaving activity. At the same DTT concentration TLCK did not inhibitMch3α but it did inhibit CPP32 activity. In contrast, at 5 mM DTTconcentration both TLCK and TPCK were unable to inhibit either Mch3α orCPP32 activity. These results indicate that the concentration of thiolagents influences significantly the activity of cysteine protease andtheir sensitivity to some inhibitors such as TLCK and TPCK.

EXAMPLE III Interrelationship of MCH3α and CPP32

This Example shows that Mch3α is a substrate for CPP32.

The ability of subunits derived from Mch3α to form active complexes withsubunits derived from CPP32 raised the possibility that Mch3α was asubstrate for CPP32 and vice versa. To test this possibility, aGST-Mch3α and GST-CPP32 fusion proteins were in vitro translated inreticulocyte lysate in the presence of ³⁵S-methionine. Briefly, Mch3α,Mch3β, GST-Mch3α and GST-CPP32 cDNAs were subcloned into the pBluescriptII KS⁺ plasmid under the T7 promoter These vectors were linearized withthe appropriate restriction enzyme and used as template for T7 RNApolymerase. The in vitro synthesized mRNA was then used for in vitrotranslation with reticulocyte lysates as described previously (Alnemriet al., supra).

To assess substrate specificity of these proteases, the labeled lysateswere incubated with recombinant active CPP32 or Mch3α enzymes (equalDEVD-AMC cleaving activity). After the Incubation period, the cleavageproducts were immobilized on GST-sepharose, washed several times andanalyzed by SDS-PAGE and autoradiography. Schematic diagrams of thevectors are shown in FIG. 3A. The results of the cleavage productsindicate that incubation of CPP32 with the in vitro translated GST-Mch3αgenerated a GST-prodomain cleavage product of molecular mass 32 kDa(FIG. 3B, Lane 5). This band was similar in size and comigrated with thebacterially expressed GST-prodomain. Although Mch3α exhibitedsignificantly less activity than CPP32 towards the in vitro translatedGST-Mch3α, a similar cleavage product was observed (FIG. 3B, lane 6).The intermediate 36 kDa minor GST-prodomain cleavage product was alsoseen in this reaction. No cleavage was observed when CPP32 was incubatedwith an in vitro translated GST control or when the in vitro translatedGST-Mch3α was incubated with buffer (FIG. 3B, lanes 2-4).

The same experiment was performed with in vitro translated GST-CPP32(FIG. 3C). In this case, CPP32 showed a very weak activity towards itsprecursor and generated a faint GST-prodomain band of 30 kDa size asexpected from cleavage at Asp28 (lane 3). No cleavage was observed inthe buffer control or the Mch3α reaction (FIG. 3C, lanes 1 and 2).Although Mch3α or CPP32 can autoactivate/autoprocess when overexpressedin bacteria, such a process is likely to be regulated in mammaliancells. Therefore, the ability of CPP32 to cleave the Mch3α precursorbetter than Mch3α itself and the weak activity of CPP32 or Mch3α towardsthe CPP32 precursor indicates that Mch3α precursor is down stream ofCPP32 and that CPP32 is likely dependent on an upstream protease foractivation in vivo.

In light of the fact that CPP32 was observed to efficiently cleave theGST-Mch3α precursor, a further purification of the cleavage productsfrom the GST-prodomain was performed and analyzed. Briefly, the³⁵S-labeled GST-Mch3α precursor was immobilized on GST-sepharose andwashed several times. The resine-GST-Mch3α precursor was incubated withactive CPP32 and the soluble products cleaved from the immobilizedGST-Mch3α precursor were then analyzed on a 10-20% gradient SDS gel andvisualized by autoradiography (FIG. 3D). The three bands that migrate as17-19 kDa proteins represent the large subunit of Mch3 at differentstages of processing. Similarly, the two bands of 12-13 kDa sizerepresent the small subunit of Mch3α. The bands that migrate as 30 and35 kDa proteins represent Mch3α precursor minus the prodomain.

In conclusion, the Mch3 gene encodes two Mch3 proteins, an active Mch3αand a Mch35 splice variant with an undetermined activity. Because of thehigh degree of homology between Mch3 and CPP32 and their ability toheterodimerize to form active heteromeric complexes, the Mch3β variantis likely to function as a dominant inhibitor of both Mch3β and CPP32.The similarity between CPP32 and Mch3α in terms of their kineticproperties and their substrate specificity towards the DEVD peptide andPARP indicates that CPP32 may not be the sole PARP cleaving enzyme inapoptosis. The possibility that Mch3α is down stream of CPP32 suggestthat CPP32 might be the PARP cleaving enzyme during the early stages ofapoptosis but that Mch3α may be involved in the final stages of PARPcleavage and apoptosis. It therefore appears that activation of thedeath program in mammalian cells is regulated by multiple pathways andthat execution of apoptosis may involve different cascades of cysteineproteases.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 17 <210> SEQ ID NO 1 <211> LENGTH: 2309<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (44)...(952) <400> SEQUENCE: 1gagagactgt gccagtccca gccgccctac cgccgtggga acg atg gc#a gat gat       55                    #                  #            Met Ala Asp As #p                    #                  #             1 cag ggc tgt att gaa gag cag ggg gtt gag ga#t tca gca aat gaa gat      103Gln Gly Cys Ile Glu Glu Gln Gly Val Glu As #p Ser Ala Asn Glu Asp 5                  #  10                 #  15                 #  20tca gtg gat gct aag cca gac cgg tcc tcg tt#t gta ccg tcc ctc ttc      151Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Ph #e Val Pro Ser Leu Phe                 25  #                 30  #                 35agt aag aag aag aaa aat gtc acc atg cga tc#c atc aag acc acc cgg      199Ser Lys Lys Lys Lys Asn Val Thr Met Arg Se #r Ile Lys Thr Thr Arg             40      #             45      #             50gac cga gtg cct aca tat cag tac aac atg aa#t ttt gaa aag ctg ggc      247Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met As #n Phe Glu Lys Leu Gly         55          #         60          #         65aaa tgc atc ata ata aac aac aag aac ttt ga#t aaa gtg aca ggt atg      295Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe As #p Lys Val Thr Gly Met     70              #     75              #     80ggc gtt cga aac gga aca gac aaa gat gcc ga#g gcg ctc ttc aag tgc      343Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Gl #u Ala Leu Phe Lys Cys 85                  # 90                  # 95                  #100ttc cga agc ctg ggt ttt gac gtg att gtc ta#t aat gac tgc tct tgt      391Phe Arg Ser Leu Gly Phe Asp Val Ile Val Ty #r Asn Asp Cys Ser Cys                105   #               110   #               115gcc aag atg caa gat ctg ctt aaa aaa gct tc#t gaa gag gac cat aca      439Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Se #r Glu Glu Asp His Thr            120       #           125       #           130aat gcc gcc tgc ttc gcc tgc atc ctc tta ag#c cat gga gaa gaa aat      487Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Se #r His Gly Glu Glu Asn        135           #       140           #       145gta att tat ggg aaa gat ggt gtc aca cca at#a aag gat ttg aca gcc      535Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Il #e Lys Asp Leu Thr Ala    150               #   155               #   160cac ttt agg ggg gat aga tgc aaa acc ctt tt#a gag aaa ccc aaa ctc      583His Phe Arg Gly Asp Arg Cys Lys Thr Leu Le #u Glu Lys Pro Lys Leu165                 1 #70                 1 #75                 1 #80ttc ttc att cag gct tgc cga ggg acc gag ct#t gat gat ggc atc cag      631Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Le #u Asp Asp Gly Ile Gln                185   #               190   #               195gcc gac tcg ggg ccc atc aat gac aca gat gc#t aat cct cga tac aag      679Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Al #a Asn Pro Arg Tyr Lys            200       #           205       #           210atc cca gtg gaa gct gac ttc ctc ttc gcc ta#t tcc acg gtt cca ggc      727Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Ty #r Ser Thr Val Pro Gly        215           #       220           #       225tat tac tcg tgg agg agc cca gga aga ggc tc#c tgg ttt gtg caa gcc      775Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Se #r Trp Phe Val Gln Ala    230               #   235               #   240ctc tgc tcc atc ctg gag gag cac gga aaa ga#c ctg gaa atc atg cag      823Leu Cys Ser Ile Leu Glu Glu His Gly Lys As #p Leu Glu Ile Met Gln245                 2 #50                 2 #55                 2 #60atc ctc acc agg gtg aat gac aga gtt gcc ag#g cac ttt gag tct cag      871Ile Leu Thr Arg Val Asn Asp Arg Val Ala Ar #g His Phe Glu Ser Gln                265   #               270   #               275tct gat gac cca cac ttc cat gag aag aag ca#g atc ccc tgt gtg gtc      919Ser Asp Asp Pro His Phe His Glu Lys Lys Gl #n Ile Pro Cys Val Val            280       #           285       #           290tcc atg ctc acc aag gaa ctc tac ttc agt ca#a tagccatatc aggggtacat    972Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gl #n         295          #       300 tctagctgag aagcaatggg tcactcatta atgaatcaca tttttttatg ct#cttgaaat   1032attcagaaat tctccaggat tttaatttca ggaaaatgta ttgattcaac ag#ggaagaaa   1092ctttctggtg ctgtcttttg ttctctgaat tttcagagac ttttttataa tg#ttattcat   1152ttggtgactg tgtaactttc tcttaagatt aattttctct ttgtatgtct gt#taccttgt   1212taatagactt aatacatgca acagaagtga cttctggaga aagctcatgg ct#gtgtccac   1272tgcaattggt ggtaacagtg gtagagtcat gtttgcactt ggcaaaaaga at#cccaatgt   1332ttgacaaaac acagccaagg ggatatttac tgctctttat tgcagaatgt gg#gtattgag   1392tgtgatttga atgatttttc attggcttag ggcagatttt catgcaaaag tt#ctcatatg   1452agttagagga gaaaaagctt aatgattctg atatgtatcc atcaggatcc ag#tctggaaa   1512acagaaacca ttctaggtgt ttcaacagag ggagtttaat acaggaaatt ga#cttacata   1572gatgataaaa gagaagccaa acagcaagaa gctgttacca cacccagggc ta#tgaggata   1632atgggaagag gtttggtttc ctgtgtccag tagtgggatc atccagagga gc#tggaacca   1692tggtgggggc tgcctagtgg gagttaggac caccaatgga ttgtggaaaa tg#gagccatg   1752acaagaacaa agccactgac tgagatggag tgagctgaga cagataagag aa#taccttgt   1812ctcacctatc ctgccctcac atcttccacc agcaccttac tgcccaggcc ta#tctggaag   1872ccacctcacc aaggaccttg gaagagcaag ggacagtgag gcaggagaag aa#caagaaat   1932ggatgtaagc ctggcccata atgtgaacat aagtaatcac taatgctcaa ca#atttatcc   1992attcaatcat ttattcattg ggttgtcaga tagtctatgt atgtgtaaaa ca#atctgttt   2052tggctttatg tgcaaaatct gttatagctt taaaatatat ctggaacttt tt#agattatt   2112ccaagcctta ttttgagtaa atatttgtta cttttagttc tataagtgag ga#agagttta   2172tggcaaagat ttttggcact ttgttttcaa gatggtgtta tcttttgaat tc#ttgataaa   2232tgactgtttt tttctgccta atagtaactg gttaaaaaac aaatgttcat at#ttattgat   2292 taaaaatgtg gttgctt              #                  #                   # 2309 <210> SEQ ID NO 2 <211> LENGTH: 303<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Met Ala Asp Asp Gln Gly Cys Ile Glu Glu Gl #n Gly Val Glu Asp Ser 1               5   #                10   #                15Ala Asn Glu Asp Ser Val Asp Ala Lys Pro As #p Arg Ser Ser Phe Val            20       #            25       #            30Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Va #l Thr Met Arg Ser Ile        35           #        40           #        45Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gl #n Tyr Asn Met Asn Phe    50               #    55               #    60Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn As #n Lys Asn Phe Asp Lys65                   #70                   #75                   #80Val Thr Gly Met Gly Val Arg Asn Gly Thr As #p Lys Asp Ala Glu Ala                85   #                90   #                95Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe As #p Val Ile Val Tyr Asn            100       #           105       #           110Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Le #u Lys Lys Ala Ser Glu        115           #       120           #       125Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cy #s Ile Leu Leu Ser His    130               #   135               #   140Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gl #y Val Thr Pro Ile Lys145                 1 #50                 1 #55                 1 #60Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cy #s Lys Thr Leu Leu Glu                165   #               170   #               175Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Ar #g Gly Thr Glu Leu Asp            180       #           185       #           190Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile As #n Asp Thr Asp Ala Asn        195           #       200           #       205Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Ph #e Leu Phe Ala Tyr Ser    210               #   215               #   220Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pr #o Gly Arg Gly Ser Trp225                 2 #30                 2 #35                 2 #40Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Gl #u His Gly Lys Asp Leu                245   #               250   #               255Glu Ile Met Gln Ile Leu Thr Arg Val Asn As #p Arg Val Ala Arg His            260       #           265       #           270Phe Glu Ser Gln Ser Asp Asp Pro His Phe Hi #s Glu Lys Lys Gln Ile        275           #       280           #       285Pro Cys Val Val Ser Met Leu Thr Lys Glu Le #u Tyr Phe Ser Gln    290               #   295               #   300 <210> SEQ ID NO 3<211> LENGTH: 2377 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (590)...(904)<400> SEQUENCE: 3gcaagctggg ctgctgggtg ggtacttcct tcaaagctga gggagcgtcc ta#cgcccacg     60cgcgcaggag ggcgcccccc gcaaagcaac gtctaggaga ccacagtgga tg#ccacagcg    120ggcccgaagc ggatcagcct tgtggatggc agatgatcag ggctgtattg aa#gagcaggg    180ggttgaggat tcagcaaatg aagattcagt ggatgctaag ccagaccggt cc#tcgtttgt    240accgtccctc ttcagtaaga agaagaaaaa tgtcaccatg cgatccatca ag#accacccg    300ggaccgagtg cctacatatc agtacaacat gaattttgaa aagctgggca aa#tgcatcat    360aataaacaac aagaactttg ataaagtgac aggtatgggc gttcgaaacg ga#acagacaa    420agatgccgag gcgctcttca agtgcttccg aagcctgggt tttgacgtga tt#gtctataa    480tgactgctct tgtgccaaga tgcaagatct gcttaaaaaa gcttctgaag ag#gaccatac    540aaatgccgcc tgcttcgcct gcatcctctt aagccatgga gaagaaaat atg# gaa tct    598                    #                  #                   #Met Glu Ser                    #                  #                   # 1 tgc tct gtc acc cag gct gga gtg cag cgg cg#t gat ctc gga aga ctg      646Cys Ser Val Thr Gln Ala Gly Val Gln Arg Ar #g Asp Leu Gly Arg Leu     5              #      10             #      15caa cct cca cct ccc agg ctt gcc gag gga cc#g agc ttg atg atg gca      694Gln Pro Pro Pro Pro Arg Leu Ala Glu Gly Pr #o Ser Leu Met Met Ala 20                  # 25                  # 30                  # 35tcc agg ccg act cgg ggc cca tca atg aca ca#g atg cta atc ctc gat      742Ser Arg Pro Thr Arg Gly Pro Ser Met Thr Gl #n Met Leu Ile Leu Asp                 40  #                 45  #                 50aca aga tcc cag tgg aag ctg act tcc tct tc#g cct att cca cgg ttc      790Thr Arg Ser Gln Trp Lys Leu Thr Ser Ser Se #r Pro Ile Pro Arg Phe             55      #             60      #             65cag gct att act cgt gga gga gcc cag gaa ga#g gct cct ggt ttg tgc      838Gln Ala Ile Thr Arg Gly Gly Ala Gln Glu Gl #u Ala Pro Gly Leu Cys         70          #         75          #         80aag ccc tct gct cca tcc tgg agg agc acg ga#a aag acc tgg aaa tca      886Lys Pro Ser Ala Pro Ser Trp Arg Ser Thr Gl #u Lys Thr Trp Lys Ser     85              #     90              #     95tgc aga tcc tca cca ggg tgaatgacag agttgccagg ca#ctttgagt             934 Cys Arg Ser Ser Pro Gly 100                 1#05 ctcagtctga tgacccacac ttccatgaga agaagcagat cccctgtgtg gt#ctccatgc    994tcaccaagga actctacttc agtcaatagc catatcaggg gtacattcta gc#tgagaagc   1054aatgggtcac tcattaatga atcacatttt tttatgctct tgaaatattc ag#aaattctc   1114caggatttta atttcaggaa aatgtattga ttcaacaggg aagaaacttt ct#ggtgctgt   1174cttttgttct ctgaattttc agagactttt ttataatgtt attcatttgg tg#actgtgta   1234actttctctt aagattaatt ttctctttgt atgtctgtta ccttgttaat ag#acttaata   1294catgcaacag aagtgacttc tggagaaagc tcatggctgt gtccactgca at#tggtggta   1354acagtggtag agtcatgttt gcacttggca aaaagaatcc caatgtttga ca#aaacacag   1414ccaaggggat atttactgct ctttattgca gaatgtgggt attgagtgtg at#ttgaatga   1474tttttcattg gcttagggca gattttcatg caaaagttct catatgagtt ag#aggagaaa   1534aagcttaatg attctgatat gtatccatca ggatccagtc tggaaaacag aa#accattct   1594aggtgtttca acagagggag tttaatacag gaaattgact tacatagatg at#aaaagaga   1654agccaaacag caagaagctg ttaccacacc cagggctatg aggataatgg ga#agaggttt   1714ggtttcctgt gtccagtagt gggatcatcc agaggagctg gaaccatggt gg#gggctgcc   1774tagtgggagt taggaccacc aatggattgt ggaaaatgga gccatgacaa ga#acaaagcc   1834actgactgag atggagtgag ctgagacaga taagagaata ccttgtctca cc#tatcctgc   1894cctcacatct tccaccagca ccttactgcc caggcctatc tggaagccac ct#caccaagg   1954accttggaag agcaagggac agtgaggcag gagaagaaca agaaatggat gt#aagcctgg   2014cccataatgt gaacataagt aatcactaat gctcaacaat ttatccattc aa#tcatttat   2074tcattgggtt gtcagatagt ctatgtatgt gtaaaacaat ctgttttggc tt#tatgtgca   2134aaatctgtta tagctttaaa atatatctgg aactttttag attattccaa gc#cttatttt   2194gagtaaatat ttgttacttt tagttctata agtgaggaag agtttatggc aa#agattttt   2254ggcactttgt tttcaagatg gtgttatctt ttgaattctt gataaatgac tg#tttttttc   2314tgcctaatag taactggtta aaaaacaaat gttcatattt attgattaaa aa#tgtggttg   2374 ctt                   #                  #                   #           2377 <210> SEQ ID NO 4 <211> LENGTH: 105<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4Met Glu Ser Cys Ser Val Thr Gln Ala Gly Va #l Gln Arg Arg Asp Leu 1               5   #                10   #                15Gly Arg Leu Gln Pro Pro Pro Pro Arg Leu Al #a Glu Gly Pro Ser Leu            20       #            25       #            30Met Met Ala Ser Arg Pro Thr Arg Gly Pro Se #r Met Thr Gln Met Leu        35           #        40           #        45Ile Leu Asp Thr Arg Ser Gln Trp Lys Leu Th #r Ser Ser Ser Pro Ile    50               #    55               #    60Pro Arg Phe Gln Ala Ile Thr Arg Gly Gly Al #a Gln Glu Glu Ala Pro65                   #70                   #75                   #80Gly Leu Cys Lys Pro Ser Ala Pro Ser Trp Ar #g Ser Thr Glu Lys Thr                85   #                90   #                95Trp Lys Ser Cys Arg Ser Ser Pro Gly             100      #           105 <210> SEQ ID NO 5 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer T50-pr1 <400> SEQUENCE: 5ccgtggaata ggcgaagag              #                  #                   # 19 <210> SEQ ID NO 6 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer SK-Zap <400> SEQUENCE: 6caggaattcg gcacgag              #                   #                  #   17 <210> SEQ ID NO 7 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer T50-pr3 <400> SEQUENCE: 7gccataaact cttcctcact t            #                  #                   #21 <210> SEQ ID NO 8 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer T50-pr4 <400> SEQUENCE: 8atggcagatg atcagggc              #                   #                  #  18 <210> SEQ ID NO 9 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer Mch3-p20 <400> SEQUENCE: 9ctagtcggcc tggatgccat c            #                  #                   #21 <210> SEQ ID NO 10 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer  Mch3-p12 <400> SEQUENCE: 10atgtcggggc ccatcaatga c            #                  #                   #21 <210> SEQ ID NO 11 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer T50-pr9 <400> SEQUENCE: 11gacccattgc ttctcagc              #                   #                  #  18 <210> SEQ ID NO 12 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Pentapeptide sequence that  #is conserved in a      number of the ICE/Ced-3 family of # proteases. <400> SEQUENCE: 12Gly Ser Trp Phe Ile  1               5 <210> SEQ ID NO 13<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Pentapeptide sequence that  #is conserved in a      number of the ICE/Ced-3 family of # proteases. <400> SEQUENCE: 13Gly Ser Trp Tyr Ile  1               5 <210> SEQ ID NO 14<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Conserved active site. <400> SEQUENCE: 14Gln Ala Cys Arg Gly  1               5 <210> SEQ ID NO 15<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Mch3 peptidyl inhibitor <400> SEQUENCE: 15Asp Glu Val Asp  1 <210> SEQ ID NO 16 <211> LENGTH: 4 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Mch3 peptidyl inhibitor <400> SEQUENCE: 16Tyr Val Ala Asp  1 <210> SEQ ID NO 17 <211> LENGTH: 4 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Mch3 peptidyl inhibitor <400> SEQUENCE: 17Asp Ser Val Asp  1

What is claimed is:
 1. An isolated antibody that specifically binds an Mch3 polypeptide said polypeptide comprising SEQ ID NO:4.
 2. The antibody of claim 1, wherein said antibody is a monoclonal antibody.
 3. The antibody of claim 1, wherein said antibody is a polyclonal antibody. 